This is a renewal application to study the mechanisms and consequences of caspase-2 activation in the stress response and in tumor suppression. While caspase-2 was the second caspase discovered, and is the most conserved caspase among the animals, its functions remain somewhat obscure. Caspase-2 can induce apoptosis, although our evidence suggests that this is indirect, requiring the cleavage of BID by caspase-2 to engage the mitochondrial pathway of apoptosis. This raises the possibility that caspase-2 may perform a primary role in some non-apoptotic function. Our studies on heat shock led us to caspase-2 when we discovered that the caspase is activated following this form of stress. We have used principles of the biochemistry of caspase activation to develop a way in which to visualize the formation of the activation platform responsible for the activation of caspase-2, and we can do this in real time by live cell imaging. Based on this and other novel approaches, and on the idea that caspase-2 can perform apoptotic and nonapoptotic roles in the cell, we will ask the following questions. How does stress activate caspase-2, leading to apoptosis? In this aim we will employ novel imaging techniques to follow the formation of the caspase-2 activation platform and activity of the enzyme following a variety of stresses. The molecular requirements for caspase-2 activation and how it is regulated will be pursued in this context. The specific relationships between caspase-2 function and engagement of apoptosis via the mitochondrial pathway of apoptosis will be studied using real-time imaging of live cells, and the requirements for caspase-2 to engage apoptosis will be examined. What are the non-apoptotic consequences of caspase-2 activation? Here, we will use clues obtained from the location of caspase-2 activation in the cell and the substrates cleaved upon activation to explore how caspase-2 affects cellular physiology under conditions that apoptosis does not occur. Our studies will focus, at least at first, on changes in the function of the Golgi upon caspase-2 activation, although other effects will also be explored. How does loss of caspase-2 promote oncogenesis? The loss of caspase-2 dramatically accelerates lymphomagenesis in the well-established E5-Myc model, and we will use this and other approaches to determine how caspase-2 functions as a tumor suppressor. We will formally test the idea that caspase-2 does this via apoptosis or by non-apoptotic effects in cells. Further, we will examine if caspase-2 acts to suppress tumor growth, or if it acts as a caretaker, such that its loss increases risk of oncogenesis by, for example, increasing genomic instability. These questions form the specific aims of our proposed research. In exploring them, we will gain fundamental insights into this orphan caspase and how its function affects cellular physiology, tumor suppression, and apoptosis. PUBLIC HEALTH RELEVANCE: Our finding that caspase-2 is activated by heat shock, combined with the recent finding that caspase-2 functions as a tumor suppressor in a murine model of lymphomagenesis, raise fundamental questions about how this caspase functions in cellular physiology. The studies proposed herein will provide mechanistic insights into these functions.